Myosin X is an unconventional myosin with a conserved motor domain followed by three IQ motifs that are potential light chain binding domains. There is a short segment predicted to form a coiled-coil that probably allows for dimerization to produce a two-headed molecule. Little is known about the function of myosin X in cells and it appears to be expressed at low abundance. In order to characterize the in vitro enzymatic properties of this myosin, we have engineered fragments corresponding to an HMM and S1 of bovine myosin X for expression in Sf9 cells. Initially, we co-expressed these fragments with calmodulin (CaM). Both constructs yielded soluble myosin containing bound calmodulin that was purified by FLAG-affinity chromatography. The myosin X-HMM-like fragment binds actin in an ATP-dependent manner and has an actin-activated MgATPase with a Vmax of 10 per sec and a Km of 5 micromolars at 37C. The MgATPase is relatively ionic strength insensitive compared to conventional myosin IIs. The myosin X-HMM translocates actin filaments in a rate of 0.18+/-0.05 microns/s in the in vitro motility assay. The transient state kinetics of myosin X S1 was also examined. These data show that myosin X binds actin more weakly in the absence of nucleotide or in the presence of nucleotide than does most myosins. In addition, its weak binding states have a higher affinity for actin than does most myosins. The rate of ADP release is about 4-5 times faster than the steady-state rate. Thus, myosin X does not appear to be a candidate for a processive vesicle transporter. Recently, we have also co-expressed the myosin X heavy chain fragments with CaM and a calmodulin-like protein (termed CLIP). The purified S1 appears to have two CaM and one CLIP bound to the heavy chain. The affinity for CLIP appears to be similar to that of calmodulin. No difference in steady-state MgATPase activities are seen in the myosin X S1 constructs with CaM alone compared to those with CaM and CLIP. We are currently characterizing the transient state kinetics of myosin X. Preliminary data suggest a slow rate of ADP binding and of ATP dissociation of acto-myosin X.